Design and Synthesis of Fluorescent Methylphenidate Analogues for a FRET-Based Assay of Synapsin III Binding

Article abstract of DOI:10.1002/cmdc.202000128

We previously described synapsin III (Syn III) as a synaptic phosphoprotein that controls dopamine release in cooperation with α-synuclein (aSyn). Moreover, we found that in Parkinson's disease (PD), Syn III also participates in aSyn aggregation and toxic

Full text of DOI:10.1002/cmdc.202000128

Accepted Article
Title: Design and synthesis of fluorescent-methylphenidate analogues
for FRET-based assay of Synapsin III binding
Authors: Andrea Casiraghi, Francesca Longhena, Valentina Straniero,
Gaia Faustini, Amy H. Newman, Arianna Bellucci, and
Ermanno Valoti
This manuscript has been accepted after peer review and appears as an
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To be cited as: ChemMedChem 10.1002/cmdc.202000128
Link to VoR: https://doi.org/10.1002/cmdc.202000128
01/2020

10.1002/cmdc.202000128
ChemMedChem
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Design and synthesis of fluorescent-methylphenidate analogues
for FRET-based assay of Synapsin III binding
Andrea Casiraghi[a,c]#, Francesca Longhena[b]#, Valentina Straniero[a]*, Gaia Faustini[b], Amy H.
Newman[c] §, Arianna Bellucci[b]*§, Ermanno Valoti[a] §
[a]
[b]
[c]
Dr. A. Casiraghi, Dr. V. Straniero, Prof. E. Valoti
Department of Pharmaceutical Sciences
University of Milan
Via Luigi Mangiagalli, 25, 20133, Milano, Italy
E-mail: andrea.casiraghi@unimi.it; valentina.straniero@unimi.it; ermanno.valoti@unimi.it
Dr. F. Longhena, Dr. G. Faustini, Prof. A. Bellucci
Department of Molecular and Translational Medicine
University of Brescia
Viale Europa, 11, 25123, Brescia, Italy
E-mail: f.longhena@unibs.it; g.faustini004@unibs.it; arianna.bellucci@unibs.it
Dr. A. H. Newman
Molecular Targets and Medications Discovery Branch
NIDA-IRP
333 Cassell Drive, 21224, Baltimore MD, United States
E-mail: anewman@intra.nida.nih.gov
*
Correspondence to: valentina.straniero@unimi.it; arianna.bellucci@unibs.it
# These authors contributed equally. § These authors contributed equally
Abstract:
histopathological hallmark of the PD brain is the presence of
abnormal intraneuronal and intraneuritic insoluble protein
aggregates called Lewy Bodies (LB) and Lewy Neurites (LN),
respectively. These are mainly composed of α-synuclein (aSyn),
We previously described Synapsin III (Syn III) as a synaptic
phosphoprotein that controls dopamine release in cooperation
with alpha-synuclein (aSyn). Moreover, we found that in
Parkinson’s disease (PD), Syn III also participates in alpha-
synuclein (aSyn) aggregation and toxicity. Our recent
a
protein enriched at synaptic terminals and governing
neurotransmitter release and recycling[3][4][5]
.
observations point to threo-methylphenidate (MPH),
a
We recently described that the synaptic phosphoprotein Synapsin
III (Syn III) interacts and cooperates with aSyn in the control of DA
release from mesencephalic neurons[6]. Moreover, we showed
that Syn III is another key component of aSyn insoluble fibrils
extracted from the post-mortem brains of sporadic PD patients[7]
and that Syn III is a crucial mediator of aSyn aggregation and
toxicity[8]. Strikingly, our most recent findings suggest that threo-
methylphenidate (MPH), a monoamine reuptake inhibitor clinically
approved for the treatment of attention deficits and hyperactivity
disorder (ADHD)[9] and for ameliorating gait freezing in advanced
PD[10][11][12][13][14],is able to stimulate an aSyn/Syn III-mediated
locomotor response, specifically in aSyn transgenic mice at a
pathological stage that exhibit severe dopaminergic functional
deficits[15]. This MPH action is lost upon in vivo Syn III gene
silencing[15], thus suggesting that this protein may act as a target
for MPH. Moreover, we observed that MPH does not stimulate
locomotion in Syn III knock out mice suggesting these actions are
monoamine reuptake inhibitor that efficiently counteracts the
freezing gait characteristic of advanced PD, as a ligand for Syn III.
Here, we designed and synthesized two different fluorescently-
labelled MPH derivatives, one with Rhodamine Red (RHOD) and
one with 5-Carboxytetramethylrhodamine (TAMRA), to be used
for assessing MPH binding to Syn III by fluorescence resonance
energy transfer (FRET). TAMRA-MPH exhibited the ideal
characteristics to be used as a FRET acceptor, as it was able to
enter into the SK-N-SH cells and could interact specifically with
human green fluorescent protein (GFP)-tagged Syn III but not with
GFP alone. Moreover, uptake of TAMRA-MPH and co-localization
with Syn III was also observed in primary mesencephalic neurons.
These findings support that MPH is a Syn III ligand and that
TAMRA-conjugated drug molecules may represent valuable tools
to study drug-ligand interactions by FRET or to detect Syn III in
cytological and histological samples.
independent of DA transporter inhibition [15]
.
Introduction
In this study, we designed, synthesized and tested two different
fluorescently-conjugated MPH derivatives to assay their ability to
interact with human Syn III by fluorescence resonance energy
transfer (FRET) in SK-N-SH neuroblastoma cells overexpressing
either green fluorescent protein (GFP) alone (as a negative
control) or human GFP-tagged Syn III.
Compounds 1 and 2 (Figure 1) were generated based on a MPH
scaffold coupled with two different fluorophores. The choice of the
fluorophores was guided by their slightly different
excitation/emission spectra, both showing good overlapping with
the spectra of the well-established FRET acceptor Red
Parkinson’s
Disease
(PD)
is
the
most
common
neurodegenerative movement disorder. The available therapeutic
options for PD are symptomatic, with no treatments capable of
halting or slowing down disease progression[1][2]. The key
neuropathological alteration in the PD brain is the loss of
dopaminergic neurons in the nigrostriatal system. The resulting
reduction of dopamine (DA) in basal ganglia circuits leads to the
onset of the classic PD-associated motor symptoms such as
rigidity, bradykinesia, tremor and postural instability. Another key
1
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10.1002/cmdc.202000128
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Fluorescent Protein (RFP). In addition, the different fluorophores
are supposed to impart different cell permeability profiles to the
assembled probes. The selected fluorophores were connected to
the MPH scaffold by two linker chains with distinct chemical
properties and comparable length. Specifically, compound 1
featured a Rhodamine Red fluorophore bound to MPH by an
amidic alkyl linker (MPH-RHOD), while compound 2 linked MPH
to 5-Carboxytetramethylrhodamine (TAMRA) by a PEG4 chain
(MPH-TAMRA).
(D,L)-threo-MPH hydrochloride is first N-protected with Boc, to
give intermediate 3, and then hydrolyzed under basic conditions
to give N-Boc-(D,L)-threo-ritalinic acid 4.
Compound 4 is the key intermediate of both the final products. In
order to obtain 2, 4 underwent esterification under Mitsunobu
reaction conditions with N-Boc-4-aminobutanol (5) and
subsequent deprotection, to give diamine 6, whose primary amine
reacts selectively with commercial Rhodamine Red-X NHS ester
to give 1.
The fluorescence intensity of MPH-RHOD, when excited by the
543 laser, was insufficient to perform FRET studies. However,
MPH-TAMRA was found to interact with human green fluorescent
protein (GFP)-conjugated Syn III but not with GFP alone in SK-N-
SH neuroblastoma cells, as detected by FRET. Moreover,
TAMRA-MPH could enter within primary mesencephalic neurons
where it co-localized with Syn III-immunopositivity.
These findings support that MPH-TAMRA can enter into cells and
can bind Syn III. Moreover, these data provide the first functional
design of a fluorescent drug probe to be used to study drug-ligand
interaction studies in cells by imaging techniques such as FRET
or fluorescence lifetime imaging (FLIM).
For the synthesis of 2, the PEG linker is introduced using
Mitsunobu reaction conditions with 4 to give N-Boc-amino-PEG4-
alcohol (7). Both the amines of 7 are then deprotected under
acidic conditions, to give 8, which is then reacted with commercial
6-COOH-TAMRA, using a TSTU-based amide coupling protocol.
The selective reaction of the NHS ester active intermediate with
the primary NH2 yields 2 as the final compound. Experimental
detail and full analytical characterization can be found under the
Chemical Synthesis section.
Results and Discussion
Establishment of TAMRA-MPH-Syn III binding by FRET
As a pivotal experiment we first analysed the red fluorescence
intensity of SK-N-SH cells overexpressing GFP-Syn III and
exposed for 15 min to either RHOD-MPH or TAMRA-MPH.
Although the RHOD-MPH-exposed cells were found to exhibit an
appreciable red signal when observed by the fluorescence
microscope, their exposure to a medium 543 laser intensity was
enough to completely quench the red fluorescence signal (Figure
2A). This supported that the fluorescence of RHOD-MPH was not
stable enough in the experimental conditions used for the FRET
experiments. Given the absence of signal observed, no
information on cell permeability and subcellular localization could
be obtained for RHOD-MPH. In contrast, the red fluorescence of
TAMRA-MPH was intense, even after exposure to a mild 543
laser intensity (Figure 2B). For these reasons, we decided not to
employ RHOD-MPH in further experiments and the presented
findings are based on TAMRA-MPH.
Figure 1: Compounds 1 and 2, object of the present work
Chemistry
In order to understand whether MPH is able to bind Syn III we
performed acceptor photo-bleaching FRET on SK-N-SH cells
transfected with a plasmid transducing GFP-human Syn III after
15 min of either TAMRA or TAMRA-MPH treatment (Figure 3).
The synthesis of 1 and 2 are described in Scheme 1.
Reagents and conditions: a) Boc2O, NaHCO3, NaCl, H2O, CHCl3, reflux (76%); b) NaOH, H2O, EtOH; c) N-Boc-4-aminobutanol, TPP, DIAD, THF, RT (85%); d) 2N
HCl/MeOH, RT (65%); e) Rhodamine Red-X NHS ester, DIPEA, DMF, 40˚C (98%); f) N-Boc-amino-PEG4-alcohol, TPP, DIAD, THF, RT (49%); g) 2N HCl/MeOH,
RT (39%); h) 6-COOH-TAMRA, TSTU, DIPEA, RT (12%).
Scheme 1. Synthesis of 1 and 2.
2
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and 100 μM) were tested on the basis of our previous findings
showing that 10 μM of MPH significantly increases aSyn/Syn III
interaction in SK-N-SH cells as detected by FRET[15]
.
We observed that cells treated with 10 μM TAMRA did not exhibit
any red fluorescent signal (Fig. 3B), supporting that the cell
permeability of this fluorophore per se is very poor. Consistently,
these cells did not display differences in the FRET efficiency when
compared to control untreated cells (Fig. 3G).
Instead, the cells exposed to increasing concentrations of
TAMRA-MPH showed a progressive increase in red fluorescence
concentration (Figure 3C-E). Interestingly, we found that, while
the cells treated with 1 μM TAMRA-MPH did not exhibit any
increase of FRET efficiency when compared to the untreated
controls; those exposed to the 10 μM concentration showed a
significant (+2.4, P < 0.001, One-way ANOVA + Newman-Keuls)
increase in the FRET signal (Fig. 3G).
Of note, the FRET efficiency in 100 µM TAMRA-MPH-treated
cultures was significantly higher than both untreated (+8.5, P <
0.001, One-way ANOVA + Newman-Keuls) and 10 µM TAMRA-
MPH treated cells (+6.1, P < 0.001, One-way ANOVA + Newman-
Keuls), indicating the occurrence of a concentration-dependent
increase in MPH-Syn III interaction (Fig. 3G).
Figure 2: Representative images showing SK-N-SH cells overexpressing GFP-
Syn III and treated with 10 μM RHOD-MPH (A) and 10 μM TAMRA-MPH (B),
respectively. As shown in panel (A), RHOD was quenched after 543 laser
exposure. Scale bars: 20 μM.
We evaluated MPH-Syn III binding by measuring FRET efficiency
between GFP and TAMRA, since TAMRA shares very similar
excitation and emission wavelengths with RFP, a well-established
FRET fluorophore when coupled with GFP[16] and we found that,
in the presence of increasing concentrations of TAMRA-MPH,
FRET efficiency was significantly higher when compared to
vehicle-treated cells. Three TAMRA-MPH concentrations (1, 10
Figure 3: Images are showing SK-N-SH cells overexpressing GFP-Syn III. Cultures were untreated (A) or treated with either 10 μM TAMRA (B), 1 μM TAMRA-
MPH (C), 10 μM TAMRA-MPH (D), 100 μM TAMRA-MPH (E), or 10 μM TAMRA-MPH + 100 μM MPH (F). Scale bars: 20 μm. The histogram (G) summarizes the
FRET efficiency in the different experimental conditions (*** P<0.001 vs. untreated; °° P<0.01 vs. TAMRA 10 μM; °°° P<0.001 vs. TAMRA 10 μM; ### P<0.001 vs.
TAMRA-MPH 1 µM; §§ P<0.01 vs. TAMRA-MPH 10 µM; §§§ P<0.01 vs. TAMRA-MPH 10 µM; ^^^ P<0.001 vs. TAMRA-MPH 100 µM; One-way ANOVA + Newman-
Keuls‘ post comparison test). The cells treated with TAMRA-MPH exhibited a significant increase of FRET efficiency starting from a concentration of 10 µM, that
was inhibited by the presence of MPH 100 µM, as shown.
3
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To corroborate that TAMRA-MPH binds to Syn III through the
MPH moiety, we performed displacement experiments by treating
SK-N-SH cells with both 10 μM TAMRA-MPH and 100 µM
untagged MPH as binding competitor (Fig. 3F). We observed that
the cells exposed to both TAMRA-MPH and untagged MPH
exhibited a significant decrease of FRET signal when compared
to those exposed to 10 μM TAMRA-MPH alone (Fig. 3G; +1.6,
P<0.01; One-way ANOVA + Newman-Keuls). This evidence
confirms that MPH binds to Syn III and supports that TAMRA-
MPH can be used as a valuable ligand to detect Syn III/MPH
interaction by FRET or other imaging methods.
In parallel, we also assessed the ability of TAMRA-MPH to bind
to GFP alone as a negative control by using GFP-transfected SK-
N-SH cells. In this case, we did not observe differences of FRET
efficiency in the vehicle-treated cells when compared to TAMRA-
MPH-treated cells (Fig. 4). This finding supports that the FRET
signal previously detected in the human GFP-Syn III-expressing
cells can be ascribed to TAMRA-MPH/Syn III interaction.
Figure 4: GFP-overexpressing SK-N-SH cells treated with TAMRA-MPH 10 μM (B), respectively. No significant differences in FRET efficiency were detected
between untreated cells (A) and cells treated with TAMRA-MPH, as shown in the graph (C) (Student’s t-test).
Evaluation of TAMRA-MPH uptake and of its co-localization
with Syn III in primary mesencephalic neurons
Indeed, we observed that GD increases the expression and
promotes the compaction of Syn III distribution in this cell culture
model[21]
.
Therefore, this in vitro cell system represents an ideal tool to
assess the ability of TAMRA-MPH to enter into neuronal cells and
to reach Syn III.
We then evaluated whether TAMRA-MPH can be internalized by
primary cultures of ventral mesencephalic neurons, as in line with
the detection of Syn III expression in caudate putamen terminals
[17][18][19]
, Syn III governs nigrostriatal neurons function[20,21]. In
Primary mesencephalic neurons were thus exposed to 1 h GD
and then subsequently treated with 100 nM TAMRA-MPH for the
following 24 hours. Paraformaldehyde (PFA)-fixed cells were the
subjected to Syn III immunolabeling and the co-localization of
TAMRA-MPH with Syn III immunopositivity was evaluated by
Airyscan super-resolution microscopy. We observed that
TAMRA-MPH signal exhibited extensive, though not complete co-
localization with the Syn III-immunopositive signal (Fig. 5A). In
particular, the co-localization appeared more abundant in
correspondence of the bigger Syn III-immunopositive dots (Fig.
5B) which are usually indicative of protein aggregates[21].These
observations further support for the conclusion that TAMRA-MPH
can be internalized by neuronal cells and bind intracellularly to
Syn III.
particular, we previously described that exposure of primary
mesencephalic neurons to insults inducing aSyn aggregation
such as glucose deprivation (GD) induces the formation of aSyn
aggregates[21,22] that also result Syn III-immunopositive[21]
.
Conclusion
In summary, the present results support that TAMRA owns the
ideal GFP-FRET couple characteristics to be exploited for
assessing drug-target interactions by this technique. Remarkably,
our results indicate that TAMRA-MPH can specifically bind Syn III
and can be used to perform FRET-based ligand-receptor
interaction studies aimed at deepening our understanding of the
pharmacodynamics profile of MPH. In line with our recent
observations supporting that Syn III is a key mediator of MPH
Figure 5: Syn III immunolabeling in primary mesencephalic neurons exposed
to GD and treated with 100 nM TAMRA-MPH (A-B). Cell nuclei were
counterstained with HOECHST. Note the presence of TAMRA red signal inside
the cells in co-localization with the green Syn III-immunopositive signal (arrows
in the merge in panel B), supporting the entrance of TAMRA-MPH in neuronal
cells and its binding to Syn III. Scale bars: 10 µm (A), 5 µm (B).
4
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action in both physiological and pathological condition[15], the
present findings constitute the first evidence demonstrating the
ability of MPH to interact with Syn III. Our previous in silico studies
showed that MPH may also bind to a specific lipid-binding aSyn
counterstained with Hoechst 33258 dye (Sigma-Aldrich), and the
coverslips were mounted onto glass slides using Vectashield (Vector
Laboratories, Burlingame, CA).
Confocal microscopy
conformation that is likely generated in advanced stages of PD [23]
.
Here, we are experimentally showing that Syn III is also a target
for MPH, that recognizes Syn III-immunopositive inclusions
generated in cells exposed to aSyn pro-aggregating stimuli.
Taken together these findings support that TAMRA-MPH could
represent a valuable imaging ligand for aSyn/Syn III co-
aggregates in PD brains and experimental models of PD. The
translational implications of our findings showing the ability of
MPH to interact with Syn III surely deserve further investigations.
Indeed, we foresee that our observations hold relevant
implications not only in the field of PD, but also of ADHD, as this
neurodevelopmental disorder has been associated with Syn III
polymorphisms[24][25][26] and is routinely treated with MPH.
For acquiring primary neuronal cells images, microscope slides were
observed by LSM 880 Zeiss confocal laser microscope equipped with
Airyscan super-resolution detector (Carl Zeiss S.p.A.) with the laser set on
λ = 405–488–543 nm and z-stack with the height of the sections scanning
= 1 μm. Images (512 × 512 pixels) were then reconstructed by Airyscan
processing and maximum intensity projection using ZEN Black Imaging
Software (Carl Zeiss S.p.A.).
Fluorescence Resonance Energy Transfer (FRET) acceptor photo-
bleaching on neuroblastoma cell line
Acceptor photo-bleaching FRET represents a useful method to study
protein-protein interaction directly into cells thanks to the application of
fluorescent tags [15]. Briefly SK-N-SH cells, a human neuroblastoma cell
line that do not express aSyn [27][28], were grown in complete medium
composed by Dulbecco's modified Eagle's medium with 1000 mg glucose/l
(Life technologies), 10% heat-inactivated fetal bovine serum, 100 μg/ml
penicillin, 100 μg/ml streptomycin and 0.01 μM non-essential amino acids
(Sigma-Aldrich). Cells were maintained at 37°C under a humidified
atmosphere of 5% CO2 and 95% O2. For FRET studies, SK-N-SH cells
were seeded onto poli-D-lysine-coated 13 mm glass coverslips in 24-well
plates (10000 cells x coverslips) and were maintained in differentiation
condition for ten days by daily adding 10 μM retinoic acid to the medium.
Seven days after seeding, cells were transiently transfected with plasmids
for the expression of Synapsin III tagged with GFP (pGFP-human Syn III)
or GFP only (pGFP-empty), by using Lipofectamine 3000 (Life
Technologies), according to the manufacturer’s instructions. Three days
after transfection, cells were treated for 15 min with vehicle (normal saline
0.9%), TAMRA or TAMRA-MPH at different increasing concentrations,
then immediately fixed with Immunofix for 15 min and subsequently
mounted on glass slides. For displacement experiments the retinoic acid
differentiated + Syn III transfected cells were treated for 15 min with both
10 μM TAMRA-MPH and 100 μM untagged threo-MPH hydrochloride
(Tocris). Once fixed, cells were analyzed by means of a Zeiss confocal
laser microscope LSM 880 (Carl Zeiss) with the laser set on λ=488–543.
The FRET efficiency (intended as the GFP acceptor recovery after TAMRA
acceptor photobleaching) was analyzed by using Zen black (Carl Zeiss).
Three-to-six regions of interest (ROI) of each double positive cell were
analyzed for 4 series and bleached after the second series with the laser
543 at 65% power. All the FRET value resulting from the different ROI were
used for statistical analysis.
Experimental Section
Animals
C57BL/6J wild type (wt) mice were used for this study. Mice were bred in
our animal house facility at the Department of Molecular and Translational
Medicine of University of Brescia, Brescia, Italy. Animals were maintained
under a 12-h light–dark cycle at a room temperature (rt) of 22 °C and had
ad libitum food and water. All experiments were made in accordance with
Directive 2010/63/EU of the European Parliament and of the Council of 22
September 2010 on the protection of animals used. All experimental
procedures conformed to the National Research Guide for the Care and
Use of Laboratory Animals and were approved by the Animal Research
Committees of the University of Brescia (Protocol Permit 719/2015-PR).
All achievements were made to minimize animal suffering and to reduce
the number of animals used.
Primary mesencephalic neuronal cell cultures and GD
Primary embryonic mouse ventral mesencephalic cells were isolated and
cultured as previously described[21]. Briefly, ventral mesencephalic tissues
were dissected from C57BL/6J wt mice at embryonic day 13.5. After
enzymatic dissociation, the single cell suspension was resuspended in
Neurobasal medium (Life Technologies) containing 100 μg/ml penicillin,
100 μg/ml streptomycin (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich)
and 2% B27 supplement (Life Technologies); cells were then centrifuged,
counted and seeded onto poly-D-lysine-coated glass coverslides in 24-
well plates for immunocytochemistry (80.000 cells/well), Cells were
maintained at 37°C under a humidified atmosphere of 5% CO2 and 95%
O2 in Neurobasal medium. After 7 days in vitro, cells were exposed to GD
in order to induce aSyn/Syn III aggregation[21,22]. GD was performed
through an incubation of the cells with Hank’s balanced salt solution
(Sigma-Aldrich) supplemented with 2 mM glutamine and 2% of B27 was
performed for 1 h at 37°C as previously described[21,22]. After GD the cells
were treated with TAMRA-MPH 100 nM for 24 h. N = 3 replicates.
Statistical Analysis
Differences between the FRET efficiency of GFP-Syn III overexpressing
SK-N-SH cells exposed to TAMRA or different concentrations of TAMRA-
MPH were assessed by One-way ANOVA followed by Newman-Keuls
multiple comparison test. Differences in terms of FRET efficiency between
GFP-overexpressing SK-N-SH cells exposed to TAMRA-MPH or not, were
assessed by using Student’s t-test. At least N = 15 cells for each
experimental condition were analyzed. In each cell 3-6 different ROIs were
acquired and the resulting median value for each cell was then plotted and
subjected to statistical analysis.
Immunocytochemistry
Twenty-four hours after TAMRA-MPH treatment, cells were fixed by
incubating for 15 min in 4% paraformaldehyde with 4% sucrose in 1 M PBS,
pH 7.4. Slides were incubated for 1 h at room temperature in blocking
solution (2% w/v bovine serum albumin (BSA, Sigma-Aldrich) plus 3% v/v
normal goat serum (Sigma-Aldrich) in PBS, then overnight at 4°C with
Synapsin III antibody (1:600, Synaptic System). On the following day, cells
were incubated for 1 h at room temperature with the 488 fluorochrome-
conjugated secondary antibody (Alexa Fluor® 488, Jackson
Immunoresearch) diluted in 0.1% Triton X-100 PBS plus BSA 1 mg/ml.
After three washes in 0.1% Triton X-100 PBS, cell nuclei were
Chemical synthesis
All starting materials were obtained from commercial suppliers and used
1
without further purification. H NMR spectra were performed at 400 MHz,
using a Varian Mercury Plus 400 instrument; the chemical shifts are
reported in ppm. Signal multiplicity is used according to the following
abbreviations: s= singlet, d= doublet, dd= doublet of doublets, t = triplet,
td= triplet of doublets, q = quadruplet, m = multiplet, sept= septuplet, and
5
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10.1002/cmdc.202000128
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bs= broad singlet. TLC was performed on standard analytical silica gel
plates (Analtech Uniplate 250 µm). Chromatographic purifications were
performed, in normal phase, using Teledyne ISCO instruments
(CombiFlash Rf or CombiFlash EZ) over different RediSep Rf disposable
chromatography cartridges. High Resolution Mass Spectrometry spectra
were acquired on LTQ-Orbitrap Velos (Thermo-Scientific, San Jose, CA)
coupled with an ESI source in positive ion mode.
threo-4-aminobutylphenidate (6): 4-(N-Boc-amino)butyl-N-Boc-threo-
phenidate (0.13 g, 0.26 mmol) was dissolved in excess 2N HCl/MeOH and
stirred at RT for 1 hour. 2.0M NH3 in isopropanol was added dropwise to
pH 9 and the solvents were evaporated under reduced pressure and the
crude product was purified by flash chromatography on silica gel. Elution
with DCM/MeOH (with 10% 8N NH4OH) 100/0 to 80/20 gave 49 mg (65%)
of threo-4-aminobutylphenidate as a colorless oil. 1H NMR (400 MHz,
CDCl3) δ 7.34 – 7.23 (m, 5H), 4.13 – 4.02 (m, 2H), 3.43 (t, J = 5.0 Hz, 1H),
3.13 (dt, J = 10.5, 2.5 Hz, 1H), 3.09 – 3.03 (m, 1H), 2.70 (dt, J = 11.9, 2.8
Hz, 1H), 2.63 – 2.57 (m, 2H), 1.72 – 1.65 (m, 2H), 1.68 (bs, 3H), 1.62 –
1.53 (m, 2H), 1.45 – 1.31 (m, 3H), 1.31 – 1.16 (m, 2H), 1.02 – 0.88 (m, 1H)
ppm.
ABBREVIATIONS
DCM: dichloromethane; DIAD: diisopropylazadicarboxyate; DIPEA:
diisopropylethylamine; DMF: dimethylformamide; mp: melting point; MW:
molecular weight; RT: room temperature; THF: tetrahydrofuran; TSTU:
N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate.
3,6-bis(dimethylamino)-9-(4-{5[4-(treo-2-pyperid-2’-yl-2-
phenylacethoxy)buthylaminocarbonyl]penthylaminosulphonyl}-2-
sulphophenyl)xanthylium, inner salt (1):
A solution of threo-4-
N-Boc-threo-methylphenidate (3): threo methylphenidate hydrochloride
(1.00 g, 3.71 mmol), sodium bicarbonate (716 mg, 8.53 mmol), sodium
chloride (867 mg, 14.8 mmol) and Boc2O (809 mg, 3.71 mmol) were
dissolved in a biphasic system of water (5 mL) and chloroform (10 mL) and
stirred at reflux for 2.5 hours. The organic phase was separated, washed
with 1N HCl (1x5 mL) and evaporated under reduced pressure. The crude
product was dissolved in hot hexane (6 mL), stirred at 0° C for 1 hour and
filtered to give 0.94 g (76%) of N-Boc threo-methylphenidate as a white
solid. mp: 81.0-84.5° C. 1H NMR (400 MHz, CD3OD): δ 7.50 – 7.41 (m,
2H), 7.38 – 7.26 (m, 3H), 4.94 – 4.82 (m, 1H), 4.26 (d, J = 10.6 Hz, 1H),
4.02 (t, J = 13.4 Hz, 1H), 3.60 (s, 3H), 3.18 – 2.98 (m, 1H), 1.74 – 1.59 (m,
2H), 1.51 (s, 9H), 1.41 – 1.28 (m, 2H), 1.26 – 1.16 (m, 2H) ppm.
aminobutylphenidate (1.89 mg, 6 μmol), Rhodamine Red-X NHS ester
(5.00 mg, 6 μmol) and DIPEA (1 μL, 6 μmol) in DMF (1 mL) was stirred at
50° C overnight. The solvent was evaporated under reduced pressure and
the crude product was purified by flash chromatography on silica gel.
Elution with DCM/MeOH (with 10% 8N NH4OH) 100/0 to 85/15 gave 6.0
mg
(98%)
of
4-N-[6-(Rhodamine
Red-4-
sulfonamido)hexanoyl]aminobutyl-threo-phenidate as a dark purple oil.1H
NMR (400 MHz, CDCl3) δ 8.81 (d, J = 1.8 Hz, 1H), 8.02 (dd, J = 7.9, 1.9
Hz, 1H), 7.30 – 7.20 (m, 9H), 7.03 (t, J = 5.6 Hz, 1H), 6.86 – 6.79 (m, 1H),
6.67 (d, J = 2.3 Hz, 1H), 4.03 (dt, J = 11.9, 6.7 Hz, 1H), 3.92 (dt, J = 11.0,
6.6 Hz, 1H), 3.56 (q, J = 7.1 Hz, 8H), 3.43 (d, J = 10.1 Hz, 1H), 3.11 (t, J =
6.6 Hz, 2H), 3.11 – 3.02 (m, 2H), 2.98 (dd, J = 12.9, 6.6 Hz, 2H), 2.67 (dt,
J = 11.7, 2.6 Hz, 1H), 2.06 (t, J = 7.5 Hz, 2H), 1.70 – 1.19 (m, 6H), 1.29 (t,
J = 7.1 Hz, 12H) ppm. HRMS: m/z 743.31, 654.22, 559.16.
N-Boc-threo- ritalinic acid (4): N-Boc threo-methylphenidate (330 mg,
1.0 mmol) and NaOH (48 mg, 1.2 mmol) were dissolved in a mixture of
water (0.75 mL) and ethanol (0.75 mL) and refluxed for 2.5 hours. The
solvents were evaporated under reduced pressure, the residue was taken
up in 5% citric acid aqueous solution (1.28 g) and ethyl acetate (1 mL) and
stirred at RT for 30 minutes. The aqueous phase was extracted with ethyl
acetate (2x1 mL), the collected organic phases were dried over MgSO4
and evaporated under reduced pressure to give 0.25 g (78%) of N-Boc
threo-ritalinic acid as a white waxy solid. 1H NMR (400 MHZ, CD3OD) δ
7.55 – 7.39 (m, 2H), 7.39 – 7.21 (m, 3H), 4.93 – 4.81 (m, 1H), 4.18 (d, J =
11.8 Hz, 1H), 4.10 – 3.92 (m, 1H), 3.19 – 3.01 (m, 1H), 1.72 – 1.59 (m,
2H), 1.50 (s, 9H), 1.42 – 1.30 (m, 2H), 1.29 – 1.08 (m, 2H) ppm.
N-Boc-amino-PEG4-alcohol: A solution of Boc2O (1.24 g, 5.69 mmol) in
EtOH (1 mL) was added dropwise to a solution of amino-PEG4-alcohol
(1.00 g, 5.17 mmol) in EtOH (9 mL) under argon atmosphere. The mixture
was stirred at RT for 4 hours. DCM (20 mL) was added and the organic
phase was washed with 1N HCl (10 mL), saturated NaHCO3 (10 mL) and
brine (10 mL). The resulting crude product was purified by filtration on a
silica gel plug, eluting at first with hexanes/ethyl acetate 70/30 and then
with DCM/MeOH (with 10% 8N NH4OH) 90/10, to give 1.12 g (74%) of N-
Boc-amino-PEG4-alcohol as a colorless oil. 1H NMR (400 MHz, CDCl3) δ
5.61 (bs, 1H), 3.77 – 3.68 (m, 4H), 3.68 – 3.59 (m, 8H), 3.55 – 3.51 (m,
2H), 3.36 – 3.27 (m, 2H), 3.01 (bs, 1H), 1.44 (s, 9H) ppm.
N-Boc-4-aminobutanol: A solution of Boc2O (3.67 g, 16.83 mmol) in DCM
(15 mL) was added dropwise to a solution of 4-aminobutan-1-ol (1.5 g,
16.83 mmol) and TEA (2.81 mL, 20.20 mmol) in DCM (10 mL). The mixture
was stirred at RT overnight. The solvent was evaporated under reduced
pressure and rotary evaporation was continued for additional 20 minutes
at 90° C. The resulting crude product was purified by filtration on a silica
gel plug, eluting at first with hexanes/ethyl acetate 60/40 and then with
DCM/MeOH (with 10% 8N NH4OH) 90/10, to give 1.21 g (38%) of N-Boc-
[N-Boc(amino-PEG4)yl] N-Boc-threo-phenidate (7): DIAD (0.30 mL,
1.55 mmol) was added dropwise to a solution of N-Boc ritalinic acid (0.33
g, 1.03 mmol), N-Boc-amino-PEG4-alcohol (0.45 g, 1.55 mmol) and
triphenylphosphine (0.41 g, 1.55 mmol) in THF (15 mL) at 0° C under an
argon atmosphere. The mixture was kept in the dark and stirred at RT for
24 hours. The solvent was evaporated under reduced pressure and the
resulting crude product was purified by flash chromatography on silica gel.
Elution with hexanes/ethyl acetate 100/0 to 60/40 gave 0.20 g (49%) of
N,N’-diBoc-threo-ritalinic acid (amino-PEG4)ester as a colorless oil. 1H
NMR (400 MHz, CDCl3) δ 7.50 – 7.40 (m, 2H), 7.37 – 7.27 (m, 3H), 5.09 –
4.79 (m, 1H), 4.30 – 4.05 (m, 4), 4.04 – 3.91 (m, 1H), 3.69 – 3.49 (m, 12H),
3.35 – 3.26 (m, 2H), 3.16 – 2.95 (m, 1H), 1.68 – 1.13 (m, 6H), 1.51 (s, 9H),
1.44 (s, 9H) ppm.
1
4-aminobutanol as a colorless oil. H NMR (400 MHz, CDCl3) δ 4.60 (bs,
1H), 3.67 (t, J = 5.4 Hz, 2H), 3.20 – 3.10 (m, 2H), 1.65 – 1.53 (m, 4H), 1.44
(s, 9H) ppm.
4-(N-Boc-amino)butyl-N-Boc-threo-phenidate (5): DIAD (92 µL, 0.47
mmol) was added dropwise to a solution of N-Boc ritalinic acid (100 mg,
0.31 mmol), N-Boc-4-aminobutan-1-ol (89 mg, 0.47 mmol) and
triphenylphosphine (123 mg, 0.47 mmol) in THF (5 mL) at 0° C under argon
atmosphere. The mixture was kept in the dark and stirred at RT overnight.
The solvent was evaporated under reduced pressure and the resulting
crude product was purified by flash chromatography on silica gel. Elution
with hexanes/ethyl acetate 100/0 to 80/20 gave 130 mg (85%) of 4-(N-
Boc-amino)butyl-N-Boc-threo-phenidate as a colorless oil. 1H NMR (400
MHz, CDCl3) δ 7.44 (d, J = 7.1 Hz, 2H), 7.37 – 7.27 (m, 3H), 5.10 – 4.98
(m, 1H), 4.91 – 4.80 (m, 1H), 4.22 – 4.09 (m, 2H), 4.04 – 3.85 (m, 2H),
3.15 – 2.94 (m, 2H), 1.68 – 1.56 (m, 3H), 1.56 – 1.46 (m, 4H), 1.51 (s, 9H),
1.44 (s, 9H), 1.33 – 1.18 (m, 3H) ppm.
(amino-PEG4)yl-threo-phenidate
(8):
N,N’-diBoc
threo-ritalinic
acid(amino-PEG4)ester (0.20 g, 0.50 mmol) was dissolved in excess 2N
HCl/MeOH and stirred at RT for 1 hour. 2.0M NH3 in isopropanol was
added dropwise until pH 9, the solvents were evaporated under reduced
pressure and the obtained crude product was purified by flash
chromatography on silica gel. Elution with DCM/MeOH (with 10% 8N
NH4OH) 100/0 to 80/20 gave 0.08 g (39%) of (amino-PEG4)yl-threo-
phenidate as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.31 – 7.21 (m,
5H), 4.30 – 4.23 (m, 1H), 4.17 (dt, J = 11.9, 4.6 Hz, 1H), 3.63 – 3.56 (m,
6H), 3.56 – 3.43 (m, 7H), 3.11 (dt, J = 10.5, 2.5 Hz, 1H), 3.08 – 3.02 (m,
1H), 2.85 (t, J = 5.2 Hz, 2H), 2.67 (dt, J = 11.9, 2.8 Hz, 1H), 1.70 – 1.63
6
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10.1002/cmdc.202000128
ChemMedChem
FULL PAPER
(m, 1H), 1.59 – 1.51 (m, 1H), 1.44 – 1.29 (m, 1H), 1.29 – 1.14 (m, 2H),
1.01 – 0.87 (m, 1H) ppm.
Acknowledgements
This work was supported by the University of Brescia Ex 60%
Research Funds to AB and by NIDA Intramural Research
Program (Z1A DA000610) during AC's year in the Newman lab.
AB is also grateful to the Michael J. Fox Foundation for
Parkinson’s Research, NY, USA, grant ID: #10742 and
#10742.01. We also thank Dr. Ludovic Muller (Structural Biology
Core, NIDA-IRP) for high resolution mass spectrometry analyses.
3,6-bis(dimethylamino)-9-({5-[treo-2-pyperid-2’-yl-2-phenylacethoxy]
(PEG4)yl aminocarbonyl}-2-carboxyphenyl)xanthylium, inner salt (2):
A solution of (amino-PEG4)yl-threo-phenidate (4.73 mg, 12 μmol), 6-
COOH-TAMRA (5.00 mg, 12 μmol) and DIPEA (2 EQ) (8.4 μL, 24 μmol)
in DMF (2 mL) was stirred at RT overnight. The solvent was evaporated
under reduced pressure and the obtained crude product was purified by
flash chromatography on silica gel. Elution with DCM/MeOH (with 10% 8N
NH4OH) 100/0 to 80/20 gave 1.2 mg (12%) of (Rhodamine Red-4-
sulfonyl)amino(PEG4)yl-threo-phenidate (Compound XVIII) as a dark
purple oil. 1H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 7.64 (s, 1H), 7.35 –
7.20 (m, 7H), 6.66 (d, J = 8.9 Hz, 2H), 6.50 (s, 1H), 6.42 (d, J = 8.5 Hz,
2H), 4.32- 4.26 (m, 1H), 4.07 – 3.99 (m, 1H), 3.71 – 3.31 (m, 14H), 3.00
(s, 6H), 3.00 (s, 6H), 2.89 – 2.79 (m, 1H), 1.94 – 1.28 (m, 6H) ppm. HRMS:
m/z 606.29, 588.24, 544.24, 472.15, 456.19, 418.15.
Conflict of interest
The authors declare no conflict of interests, financial or otherwise,
in this paper.
Keywords: fluorescent-methylphenidate, TAMRA, FRET,
Synapsin III binding, Parkinson’s Disease.
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10.1002/cmdc.202000128
ChemMedChem
FULL PAPER
Table of Contents
The present study presents TAMRA-threo-methylphenidate as a promising tool for the study of drug-ligand interactions.
We designed and synthetized this fluorescent conjugate and we assessed its ability to interact with human Synapsin III, a synaptic
partner of alpha-synuclein co-participating in Parkinson’s disease pathology, by FRET.
8
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